1. Field of Invention
This invention is related to amyloid β proteins and detection, prevention and treatment of Alzheimer's disease.
2. Background of the Prior Art
Alzheimer's disease is a progressive neurodegenerative disease, characterized by distinct pathologies, including neurofibrillary tangles, neuritic plaques, neuronal atrophy, dendritic pruning and neuronal death. Neurofibrillary tangles represent the collapsed cytoskeleton of dead and dying neurons, while neuritic plaques are extracellular deposits of various protein, lipid, carbohydrate and salt components, the primary protein component of which is a 39-43 residue peptide known as amyloid β. Historically, it is these pathologic hallmarks that have defined Alzheimer's disease, rather than a distinct cellular or physiologic mechanism. Undoubtedly, there are specific operative mechanisms responsible for most of what we currently call Alzheimer's disease; however, these mechanisms very likely operate within a background environment that variously affords some level of protection, or exerts contributing and exacerbating effects. The result is a very broad age/incidence distribution curve, with few clues from population studies that point to specific causes.
One of the significant recent successes has been the identification of specific mutations in proteins on chromosome 14 (Sherington et al., 1995) and chromosome 1 (Levy-Lahad et al., 1995) that are linked to hereditary Alzheimer's disease, augmenting earlier findings involving two different sites of mutations in the APP gene. All of these mutations have now been shown to converge into a final common causative pathway for Alzheimer's disease that involves elevated levels of amyloid β-42/3, the long form of Aβ that is found prevalently in AD plaques because it is highly prone to rapid aggregation in aqueous media.
Amyloid β in Alzheimer's Disease. The molecular era in Alzheimer's research dates to 1984, when Glenner and Wong (1984a) succeeded in isolating and identifying the cerebrovascular amyloid associated with Alzheimer's disease. Subsequently, Glenner and Wong (1984b) and Masters et. al. (1985ab) identified the same 39-43 residue peptides now known as amyloid β, as the major protein component of Alzheimer's disease neuritic plaques. This represented the first time a discrete molecule had been linked to Alzheimer's disease, a disease which to that point had been characterized only by neuroanatomy and neuropathology descriptions. Amyloid β also was identified as the plaque component in brains of Down's Syndrome individuals, (Glenner and Wong 1984b, Masters 1984b), leading to the suggestion that the gene encoding it might exist on chromosome 21. By 1987, a number of groups had used the amyloid β sequence information and molecular genetics techniques to validate that suggestion, identifying the gene for the amyloid precursor protein (APP). (Kang et al., 1987, Tanzi et al., 1987).
The APP gene is a large, multi-exon gene that is differentially spliced into a number of APP's (reviewed in Selkoe, 1994). The proteins are large transmembrane proteins, now known to be processed by several pathways, one or more of which may generate amyloid β. The earliest studies of APP processing had suggested that amyloid β formation was not a normal process (Esch et al., 1990, Sisodia et al., 1990), though subsequent studies in cultured cells and analysis of serum and cerebrospinal fluid have shown that amyloid β formation occurs as a normal process in many cell types, though its formation may not represent a predominant overall pathway.
Pivotal genetic studies of DNA from individuals afflicted with early onset of familial Alzheimer's disease revealed that mutations in a single gene, this same APP gene, were causative for this very severe form of the disease. Interestingly, several different mutations in the APP gene were found including three different single residue substitutions at Val 717, four residues downstream of the amyloid β 1-42 C-terminus (Goate et al., 1991, Chartier-Harlan, et al., 1991, Murrell, et al., 1991), and a two residue mutation (670-671) immediately upstream of the amyloid β N-terminus, associated with early onset familial Alzheimer's disease in a Swedish family (Mullan, et al., 1992). When a vector encoding the cDNA of the Swedish mutant APP gene was transfected into cell lines to evaluate APP processing, it was found that six-eight times more amyloid β was formed, when compared with levels from wild-type APP (Citron, et al., 1992, Cai et al., 1993). It has also demonstrated that brain tissue extracts containing native human brain protease activities were able to process a fluorogenic octapeptide substrate encompassing the Swedish mutation more than 100-fold faster than the corresponding substrate based on the wild-type sequence (Ladror, et al., 1994). These results suggest that the mechanism by which the Swedish mutation causes early onset familial Alzheimer's disease involves substantial overproduction of amyloid β. Similar studies of amyloid formation in cells transfected with the 717 mutant APP also had been conducted, but the levels of amyloid β produced were not different from levels produced by wild-type APP. This led to mechanistic speculations that something other than amyloid β production was pathogenic for these mutations. A closer evaluation of processing of the APP 717 mutant, and the Swedish mutant APP by Younkin and co-workers (Suzuki et al., 1994) proved to be pivotal in providing a unified picture of these genetic Alzheimer's disease cases. In this study, not only were total levels of amyloid β production evaluated, but the specific lengths of the amyloid β peptides produced were also analyzed. The results indicated that the 717 mutation led to more than a doubling of the ratio of amyloid β 1-42 to amyloid β 1-40 (a highly soluble peptide under physiologic conditions) even though total amyloid β levels did not change. The recently discovered presenilin 1 and 2 familial Alzheimer's disease mutations in genes residing on chromosome 14 and chromosome 1, respectively, have also been linked to significant overproduction of amyloid β 1-42. (Mann et al., 1996, Schuener et al., 1996) Based on these findings, it is reasonable to theorize that the pathogenic process meditated by these distinctly different familial Alzheimer's disease mutations is the production of greater levels of amyloid β 1-42, the form of amyloid that aggregates most readily (Snyder et al., 1994), and the form that appears to seed aggregation of amyloid β to form neuritic plaques (Roher et al., 1993, Tamaoka et al., 1994).
Non-amyloid Plaque Components in Alzheimer's Disease Amyloid β is the major protein component of plaques, comprising more than 70% of the total protein. A variety of other protein components also are present, however, including α1-antichymotrypsin (ACT), heparin sulfate proteoglycans (HSPG), apolipoproteins E and J, butyrylcholinesterase (BChE), S-100B, and several complement components. While the importance of these components in the onset and progression of Alzheimer's disease has not been established, involvement of apo E isoforms in the disease has been established by genetic studies of Roses and colleagues (Strittmatter et al., 1993), who discovered that a polymorphism in the apolipoprotein E gene, namely apo E4, correlated with earlier onset of Alzheimer's disease in a large set of late-onset familial Alzheimer's disease cases. Subsequent studies have confirmed that groups of individuals with apo E4 have a significantly greater risk of Alzheimer's disease and that the onset of Alzheimer's disease roughly parallels the gene dosage for apo E4. On a mechanistic level, studies have revealed that apo E4 binds with lower affinity to amyloid β than apo E3 or apo E2, isoforms which are associated with later onset of Alzheimer's disease. It has been suggested that these isoforms may exert a protective effect by more effective clearance of amyloid β 1-41 deposits (LaDu et al., 1994, 1995).
The role of other plaque components is not as clear, though recent studies (Oda et al., 1995) have shown that apo J (clusterin) can significantly enhance the toxicity of aggregated amyloid β 1-42 in vitro. It also has been reported that HSPG enhances the toxicity of amyloid β 1-40 when injected into rat brain (Snow et al., 1992). Wright et al. (1993) demonstrated that amyloid plaques from Alzheimer's disease brain contain significant levels of BChE, while amyloid plaques from elderly non-demented individuals do not. The acute phase inflammatory protein ACT also is upregulated in Alzheimer's disease brain, and it is known to associate with the N-terminal 16 residues of amyloid β. Ma et al. have reported that ACT can enhance the aggregation of amyloid β 1-42, and these authors speculate that the enhanced aggregation contributes to its neurotoxicity.
Amyloid β Cellular Responses and In Vivo Pathology. Beyond the plaques and tangles that are the hallmarks of Alzheimer's disease, it is clear that a range of cellular responses has been induced, both in neurons and in accompanying glial cells. At a biochemical level, hyperphosphorylation of the tau protein is evident, resulting from perturbation of the kinase/phosphatase balance. At a transcriptional level, a variety of genes is activated to produce a spectrum of proteins not normally present or only present at lower levels in the brain. There also is significant evidence that inflammatory processes have been activated.
In spite of the large volume of knowledge accumulated from neuropathological and immunohistochemical studies, it is difficult to distinguish which events are associated with early, causative processes, and which are simply late stage phenomena. Presently, relatively few specific signaling processes or responses that occur in cell culture have been documented to occur in vivo, and few molecular hallmarks from Alzheimer's disease brain tissue have been shown to be activated in cultured neurons subjected to a range of possible degenerative insults likely to be involved in Alzheimer's disease. One of these processes, tau phosphorylation, has been documented to be induced by aggregated amyloid β1-42 in differentiated SH-SY5Y cells (Lambert, et al., 1994), and this result has been confirmed in a more recent report by Busciglio et al. (1995), in which amyloid β activated tau phosphorylation in cultured primary rat hippocampal neurons.
Fibrillar Amyloid β and Neurodegeneration in Alzheimer's Disease. The precise and detailed mechanism by which amyloid β 1-42 causes Alzheimer's disease has not been elucidated, but the literature contains more than 200 studies of amyloid β neurotoxicity, many of which have been reviewed recently (Yankner, 1996; Iversen et al., 1995). The consensus view that has emerged in the literature is that in order for amyloid β to be toxic, it must assemble into fibrillar structures (Pike et al., 1993). Solutions containing only monomeric amyloid β have repeatedly been demonstrated to have no deleterious effect on neurons in culture. Furthermore, studies apparently have been able to correlate the formation of amyloid β-sheet containing fibrils and the timing and extent of toxicity using techniques such as circular dichroism and electron microscopy (Simmons, et al., 1994). One study concluded explicitly that amyloid β must exist in fibrillar form in order for it to be toxic (Lorenzo and Yankner, 1994). Despite this consensus regarding amyloid β structure and activity, there continues to be a problem of reproducibility of published experimental work involving amyloid toxicity, and widespread variability of activity obtained with different batches of amyloid, or even the same batch of amyloid handled in slightly different ways, in spite of identical chemical composition (May et al., 1992). This has raised questions regarding the precise structures of amyloid β that are responsible for its activity.